A fuel cell is an electric cell that converts the chemical energy of a fuel directly into electric energy in a continuous process. The efficiency of this conversion can be made much greater than that obtainable by thermal-power conversion. In the latter the chemical reaction is made to produce heat by combustion. The heat is then transformed partially into mechanical energy by a heat engine, which drives a generator to produce direct current.
Although, in principle, the nature of the reactants is not limited, the fuel-cell reaction usually involves the combination of hydrogen with oxygen, as shown by Equation (1). At 25.degree. C. and 1 atmosphere pressure, that is, standard temperature and pressure (STP), the reaction takes place with a free energy change (.DELTA.G) of .DELTA. G = 056.69 kcal/mole, that is, 237,000 joules/mole water. EQU H.sub.2 (g) + 1/2O.sub.2 (g) .fwdarw. H.sub.2 O(/) (1)
if the reaction is harnessed in a galvanic cell working at 100% efficiency, a cell voltage of 1.23 volts results. In actual service such cells have shown steady-state potentials in the range 0.9-1.1 volts, with reported coulombic efficiencies of the order 73-90%.
Fuel cells are of 200-500 watts capacity and 50-100 ma/cm.sup.2 current density. Larger prototypes have been produced, some as large as 15 kw capacity.
The most successful prior art type is the H.sub.2 --O.sub.2 fuel cell of the direct or indirect type. In the direct type, hydrogen and oxygen are used as such, the fuel being produced in independent installations. The indirect type, employes a hydrogen-generating unit which can use as raw material a wide variety of fuel. The reaction taking place at the anode is as in Eq. (2), and at the cathode as in Eq. (3). EQU 2H.sub.2 + 4OH- .fwdarw. 4H.sub.2 O + 4e- (2) EQU O.sub.2 + 2H.sub.2 O + 4e .fwdarw. 4OH- (3)
because of the low solubility of H.sub.2 and O.sub.2 in electrolytes, the reactions take place at the interface electrode-electrolyte, requiring a large area of contact for a large electron flow. This is obtained with porous materials called upon to fulfill the following main duties: The materials must provide contact between electrolyte and gas over a large area, catalyze the reaction, maintain the electrolyte in a very thin layer on the surface of the electrode, and act as leads for the transmission of electrons.
Sun powered photosynthetically-driven biological fuel cells are also known to the prior art. In U.S. Pat. No. 3,477,879, a device is described in which an electrical fuel cell is formed by using two chambers, one placed in sunlight and supplied with nutrients and microorganisms which transfer light energy or photons into chemical energy in the form of algae or carbohydrate, and the other placed in the dark where the chemical energy is released by reducing bacteria which produces compounds which release electrons. A bridge is included in the device to provide a pathway for cations and anions without a transfer of material between chambers. Electrons are released to an anode of the device by sulfites generated from sulfates by bacterial action. The energy of this action is derived from the sun and stored as bacterial metabolites, these being fed to the bacteria to drive the reduction reactions generating compounds which, in turn, give up electrons to an electrode element.
The optimal condition for a photosynthetically-driven fuel cell would be one in which the cells collecting sunlight had as their genetic-based biochemical directive that most of the photonic energy captured within the chloroplasts of the cells (assuming eucaryotic cells are used rather than photosynthetic bacteria or blue-green algae) would be exported from within the living cells to outside of the cell organism, where it could be acted upon without further catabolism by any other organism to produce electrons with a negative standard reduction potential as close as possible to hydrogen (-0.42 volts).
Work with chloroplast preparations has shown that it is possible to produce molecular hydrogen with certain enzymes in the absence of appreciable oxygen tensions from sunlight.
Oxygen (+0.82 volts) produced by the water-splitting activity of photosynthesis would consititute a readily-available source of oxidant, and should be thought of as the oxidant of choice for accepting electrons at the cathode, whether the cathode is separated from the living cells to which oxygen is delivered, or is spatially set among the cells to which oxygen diffuses.
In photosynthesis, four photons captured by a chlorophyll pigment system with an average energy of approximately 50 Kcals per einstein are needed to reduce one molecule of nicotinamide adenine dinucleotide phosphate (NADPH) at approximately 53 Kcals per mole. Therefore, the theoretical maximum conversion of photonic energy to reducing potential is approximately 25%. Tapping the energy as formed into carbohydrate leads to another reduction in the theoretical efficiency. The actual efficiency of photosynthesis in nature for recovered energy in fixed carbon for a field of sugar cane, one of the most efficient species, is as high as eight percent.
A cell type which approaches these model characteristics was first described in the prior art as a type of cell living within the leaves of certain highly photosynthetically-efficient tropical plants such as crab grass or sugar cane. In the field of plant physiology, what is termed Kranz-type leaf anatomy has been described in the prior art in which the vascular bundles are surrounded by two concentric chlorophyllous layers, thereby forming an inner parenchyma bundle sheath layer and an outer mesophyll layer. It has also been found that these species with Kranz-type leaf anatomy also fix carbon dioxide into four carbon compounds such as oxaloacetate, aspartate or malate, rather than by Calvin cycle type CO.sub.2 condensation with ribulose bisphosphate. These species are described as "C.sub.4 " type plant species. "C.sub.4 " plant species are further segregated into what are termed "malate formers" or "aspartate formers" depending upon which compound appears to be the major immediate product of carbon fixation.
It was also noted in the prior art that this newly-discovered mode of carbon dioxide formation was associated with Kranz-type leaf anatomy. Further investigation revealed that the mesophyll cell type was responsible for the carbon dioxide fixation reaction, and that this cell was thought to transport carbon dioxide and reducing equivalent derived from the sun to the neighboring bundle sheath cell type. The mesophyll cells collect carbon dioxide and sunlight, while the bundle sheath cells specialize in carbohydrate formation from the carbon dioxide and energy in malate which is transported to the bundle sheath cells. Another prior art finding is that mesophyll cells from C.sub.4 species have reduced photo-respiration rates which, in other plant species, use up energy and reduce the overall conversion efficiency of the energy in photons to energy in chemical bonds. The C.sub.4 plants also are more efficient users of solar energy at high light intensities than plant species using the first discovered means of carbon dioxide fixation, such as spinach.
Recent investigations in the field of plant physiology have demonstrated that mesophyll cells, from what are termed "C.sub.4 malate formers" in the scientific literature, have the possibility of producing extracellular reducing equivalents at the level of malate which can be transported through a series of oxidation and reduction reactions to an inert electrode. A suspension of mesophyll cells isolated from the leaves of these species export malate and absorb pyruvate and CO.sub.2. The oxygen (+0.82 volts) produced from the water-splitting activity in photosynthesis can be used to accept electrons at a cathode.
Within "C.sub.4 " type mesophyll cells, the NADPH formed in the chlorplast is used to reduce oxaloacetate to malate which is transported to bundle sheath cells which in nature lie next to mesophyll cells in the leaf. By eliminating the bundle sheath cells and using malic enzyme, malate can be oxidatively decarboxylated to pyruvate which is then shuttled back to the mesophyll cell and serves as a precursor of phosphoenol pyruvate production and, hence, malate formation with the mesophyll cell. Thus, we are able to interdict the normal flow or reducing equivalents at the NADPH (-0.32 volts) level and use the energy to produce a current of electons. By the use of malic enzymes (L-malate: NAD.sup.+ or NADP.sup.+ oxidoreductase (decarboxylating) EC 1.1.1.38 or EC 1.1.1.40), the extracellular malate can be converted to CO.sub.2 and pyruvate as two electrons are transferred to reduce a nicotinamide adenine dinucleotide [NAD((P)H].
The technology for transferring electrons from extracellular NADPH to the electrode element of a fuel cell has been demonstrated in the prior art, using a potential mediator substance (benzyl viologen) to transfer electrons from NADPH to an electrode to measure the standard reduction potential of the NADPH/NADP+ couple. It was found that xanthine oxidase was necessary to catalyze the reaction. Other workers in the art have used other flavoprotein NADH and NADPH dehydrogenases to catalyze the transfer of electrons from these two compounds (NADH and NADPH) through various reducible dye intermediates and other reducible compounds, such as quinone, which serve as potential mediator compounds for delivery of electrons to an inert electrode.
Methyl phenazonium methosulphate and similar compounds have also been used to transfer electrons from the nicotinamide adenine dinucleotide and the phosphate analog, NADPH, directly to a fuel cell electrode without the necessity of an intervening enzyme step. Malate coming from C.sub.4 mesophyll cells in some way must be made to give up electrons and form pyruvic acid (as in the case of malate, pyruvic acid is the acid form of pyruvate; the form is dependent upon the hydrogen ion concentration of the surrounding environment), which is reabsorbed by mesophyl-cells and serves as a precursor to malate formation within the mesophyll cell. Malic enzymes ((L-malate: NAD.sup.+ oxidoreductase (decarboxylating) EC 1.1.1.38 and EC 1.1.1.40) are known to exist in multiple forms. It can be isolated from bundle sheath cells from C.sub.4 type plants, lactobacillus bacteria, cactus, or other species. Malic enzyme catalyzes the following reversible reactions: ##EQU1##
The equilibrium constant favors malate formation when CO.sub.2 is at a pressure of 760 mm of Hg and is 5.times.10.sup.-2 mole.sup.-1. Pyruvate and CO.sub.2 are taken up by mesophyll cells, and NAD.sup.+ or NADP.sup.+ is reformed as NADH or NADPH gives up electrons which find their way to an electrode; thus, the reaction can be made to go toward malate oxidation as the products of the reaction are removed as current flows through the circuit. As previously mentioned, there are several methods to transfer electrons from the nicotinamide adenine dinucleotides to the fuel cell electrode. An important consideration in the transport of the two electrons from NADH or NADPH to an electrode assembly is that the xanthine oxidase catalyze step as well as the donation of electrons step at the surface of the electron accepting electrode assembly must be conducted in the absence of high oxygen tension in the aqueous solution because oxygen can serve as the electron acceptor at both of these reaction surfaces. Methyl phenazonium methosulphate is also susceptible to oxidation by molecular oxygen.
Tissue culture technology has progressed so that the means of growing and maintaining cultures of cells of the mesophyll cell types has evolved. When furnished with the right concentrations of inorganic salts and organic growth-stimulating subtances, mesophyll cells have been kept autotrophic for extended periods as described in the prior art.
Enzymes used in the biological fuel cells under consideration must be kept from proteolytic attack by bacterial agents contaminating the suspension of living mesophyll cells for an extended function of the device. This may be achieved by using polyacrylamide cross-linked polymers or other polymeric compounds capable of forming a clathrate type molecular cage around the protein enzyme. Covalent linkage of the malic enzyme to the lattice structure may or may not be necessary depending upon compounds and techniques employed. If a second enzyme is employed to transfer electrons from the reduced nicotinamide adenine dinucleotide to a potential mediator substance, this protein also should be enclosed within a protective element.
The mesophyll cells can be isolated from what are termed C.sub.4 malate-forming species of plants by either enzymatic methods, as described by Jense, Plant Physiol. 48 9-13, 1971, or Gnanam and Kulandaivelu, Plant Physiology 44: 1451-1456, 1969, and adapted toward separating C.sub.4 mesophyll cells by various plant physiologists or grinding techniques as employed by Edwards and Black, Plant Physiology 47: 149-156, 1971, can be used to free mesophyll cells from other cells of the leaf such as bundle sheath cells. Single mesophyll cells of Digitaria sanguinalis are approximately 15-25 u meters in diameter and can be separated from the other cell types by passage of solution containing the cells through nylon filtration net, of approx. 30 u meters pore size, which passes mesophyll cells but not bundle sheath cells and bundle sheath strands of cells. Mesophyll cells can be separated from chloroplasts and broken cell fragments and most bacteria by catching the cells on a 10 u meters net. Further purification of mesophyll cell cultures and the development of a culture from a single mesophyll cell used to clone the mesophyll cells used for the invention is within the technology. Best results are achieved when cells are isolated from the leaves of plants with seeds surface sterilized in 1.8% NaClO.sub.4 (sodium hypochlorite, bleach) for 10-15 minutes and germinated and grown on sterile agar containing essentials salts such as Hogalands salts, or commercial hydroponic garden salts.
The present invention is concerned with an improved type of photosynthetically-driven biological fuel cell. The invention relates specifically to a biological fuel cell for transducing the energy in sunlight quanta into a useable direct electric current. This is achieved by the action of sunlight upon living cells whose genetic makeup and differentiation dictate that the cells will export to the extracellular space a large fraction of the reducing equivalents generated by sunlight in the chloroplasts of the cells. The fuel cell of the invention uses photosynthetic cells isolated from the leaves of Digitaria sanguinalis (crabgrass); however, mesophyll cells from any "C.sub.4 " type photosynthetic plant species may be used in which malate (malic acid) serves as the medium of transfer of reducing equivalents between mesophyll cells and naturally occurring neighbor cells (called bundle sheath cells).